CN112831503B

CN112831503B - Rice sheath blight resistance gene SBR11 and molecular marker and application thereof

Info

Publication number: CN112831503B
Application number: CN202110241489.1A
Authority: CN
Inventors: 王雨; 左示敏; 薛芗; 陈宗祥; 冯志明; 胡珂鸣; 潘学彪
Original assignee: Yangzhou University
Current assignee: Yangzhou University
Priority date: 2021-03-04
Filing date: 2021-03-04
Publication date: 2022-11-18
Anticipated expiration: 2041-03-04
Also published as: CN112831503A

Abstract

The invention discloses a rice sheath blight resistance gene SBR11, and a molecular marker and application thereof, and belongs to the technical field of crop molecular biology. The resistance of SBR11 gene to banded sclerotial blight is proved through genetic transformation, the functional variation of the gene caused by SNP at sites 190 and 926 of a coding region of the gene is proved through allele function and haplotype analysis, and two molecular markers for detecting the genotypes of the two sites are further developed, wherein the molecular markers comprise the SNP site 190 and the SNP site 926, and the molecular markers can be used for detecting the allele of SBR11 and selecting, so that more resources are provided for designing and breeding rice disease-resistant molecules, and the breeding process is accelerated.

Description

Rice sheath blight resistance gene SBR11 and molecular marker and application thereof

Technical Field

The invention belongs to the technical field of crop molecular biology, and relates to a rice sheath blight resistance gene SBR11, and a molecular marker and application thereof.

Background

Rice (Oryza sativa L.) is the most important food crop in China. The rice sheath blight disease caused by the strong saprophytic fungus Rhizoctonia solani (Rhizoctonia solani Kuhn) is one of three major diseases of rice in China. The sheath blight disease has wide occurrence area and great control difficulty, so the yield loss is positioned at the head of each disease of rice. For a long time, the prevention and treatment of sheath blight mainly depends on chemical agents, but the effect is always poor. The popularization and the cultivation of disease-resistant varieties are the most economic and effective measures for controlling diseases. The resistance of rice to sheath blight belongs to Quantitative character resistance, is controlled by polygene or QTL (Quantitative trait Locus), and has important theoretical value and practical significance for cloning related genes for resisting sheath blight.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a rice sheath blight resistance gene SBR11 as well as a molecular marker and application thereof, and the invention proves that the resistance of the SBR11 gene to sheath blight is positively regulated through genetic transformation. Through allele function and haplotype analysis, the SNP at the 190 and 926 sites of the coding region of the gene is proved to cause gene function variation, and further, the invention also develops two molecular markers for detecting the genotypes of the two sites.

The technical scheme of the invention is as follows:

one of the purposes of the invention is to provide a rice sheath blight resistance gene SBR11, which is any one of the following genes:

(1) The full-length sequence of the SBR11 is shown as SEQ ID NO. 1; or

(2) The nucleotide sequence shown in SEQ ID NO.1 is substituted, deleted and/or added with one or more genes of which the nucleotides encode the same functional protein; or

(3) The 190 th base of the SBR11 with the full-length sequence of SEQ ID NO.1 is changed from T to A; or

(4) The full-length sequence of the SBR11 is that the 926 nd base of the sequence shown as SEQ ID NO.1 is changed from G to A; or

(5) The full-length sequence of the SBR11 is that the 1492 nd base of the sequence shown by SEQ ID NO.1 is changed from A to G.

The other object of the present invention is to provide the protein encoded by the rice sheath blight resistance gene SBR11, wherein the amino acid sequence of the protein comprises:

(1) An amino acid sequence shown as SEQ ID NO. 2; or

(2) Protein which is derived from the protein 2) and has the same activity and is obtained by substituting, deleting and/or adding one or more amino acids in the amino acid sequence shown in SEQ ID NO. 2.

The invention also aims to provide a biological material containing the rice sheath blight-resistant gene SBR11, wherein the biological material is a vector, a transgenic cell line, an engineering bacterium, a host cell or an expression cassette.

The fourth purpose of the invention is to provide the application of the rice sheath blight resistance gene SBR11, protein or biological material in rice sheath blight resistance breeding.

The fifth purpose of the invention is to provide a molecular marker for detecting the rice sheath blight resistance gene SBR11, wherein the molecular marker comprises an SNP site 190 and an SNP site 926, the SNP site 190 is positioned at the 190 th base of a coding region of the SBR11, the base is subjected to SNP variation, and the polymorphism is T/A; the SNP site 926 is located at the 926 nd base of the SBR11 coding region, and the polymorphism of the SNP site is G/A.

In the molecular marker, the SNP locus 190 is located at 4851244 th base of No. 11 chromosome of rice, 176bp sequences before and after the SNP locus 190 are shown as SEQ ID No.3, and the 154bp base is the SNP locus 19; the SNP locus 926 is located at 4851980 th base of No. 11 chromosome of rice, the sequence of 262bp before and after the SNP locus 926 is shown as SEQ ID No.4, and the 125bp base is the SNP locus 926.

The sixth object of the present invention is to provide a primer combination for detecting the above molecular marker, which comprises a primer set for detecting SNP site 190 and SNP site 926; primers for detecting the SNP site 190 are Primer190F and Primer190R, wherein the sequence of the Primer190F is shown as SEQ ID NO.5, and the sequence of the Primer190R is shown as SEQ ID NO. 6; the primers for detecting the SNP site 926 are Primer926F and Primer926R, wherein the sequence of the Primer926F is shown as SEQ ID NO.7, and the sequence of the Primer926R is shown as SEQ ID NO. 8.

The seventh purpose of the invention is to provide a method for detecting the rice sheath blight-resistant gene SBR 11:

carrying out PCR amplification on sample genome DNA by primers with nucleotide sequences shown as SEQ ID NO.5 and SEQ ID NO.6, carrying out enzyme digestion on the PCR amplification product by using restriction endonuclease PvuII, and detecting the enzyme digestion product in agarose gel electrophoresis with the concentration of 4%; if a characteristic band of 176bp appears in the electrophoresis result, the base of the rice to be detected at the SNP190 position is consistent with the SBR11 disease-resistant allele SBR 11; if a characteristic band of 155bp appears in the electrophoresis result, the base of the rice to be detected at the SNP190 position is consistent with the SBR11 disease-sensitive allele SBR 11;

carrying out PCR amplification on sample genome DNA by primers with nucleotide sequences shown as SEQ ID NO.7 and SEQ ID NO.8, carrying out enzyme digestion on the PCR amplification product by using restriction enzyme DdeI, and detecting the enzyme digestion product in agarose gel electrophoresis with the concentration of 4%; if two characteristic bands of 124bp and 140bp respectively appear in the electrophoresis result, the base of the rice to be detected at the SNP926 position is consistent with the SBR11 disease-resistant allele; if a 262bp characteristic band appears, the base of the rice to be detected at the SNP190 position is consistent with the SBR11 susceptible allele SBR 11;

only if the genome of this sample is identical to SBR11 disease-resistant allele SBR11 at SNP190 and SNP926, it can be judged that this sample contains SBR11 disease-resistant allele SBR11, otherwise this sample contains SBR11 susceptible allele SBR11.

The eighth purpose of the invention is to provide the application of the molecular marker, the primer combination or the detection method in the detection of the rice sheath blight resistance gene SBR11.

The invention has the following beneficial effects: the invention clones the rice sheath blight resistance gene SBR11, and verifies the function of the gene through genetic transformation: the expression level of the SBR11 gene is enhanced by using an overexpression technology, and the expression level of the SBR11 gene is reduced by using an RNAi technology, so that the gene can obviously improve the resistance of rice to sheath blight. This study also analyzed the type of susceptible allelic variation of SBR11 and developed functional markers for the identification of disease resistance alleles. The marker can be used for detecting and selecting the SBR11 allele, provides more resources for the design and breeding of rice disease-resistant molecules and accelerates the breeding process.

Drawings

FIG. 1 shows the results of field resistance identification of SBR11 overexpression material (SBR 11 OE);

FIG. 2 shows the results of field resistance identification of SBR11 interfering material (SBR 11 RNAi);

FIG. 3 shows the results of field resistance identification of SBR11 complementary material (SBR 11 CP);

FIG. 4 is a schematic diagram of the sequence variations of the SBR11 promoter and coding region in Teq (TQ) and Lemont (LE);

FIG. 5 shows the expression levels of SBR11 in the case of LE and NIL after inoculation of stringy blight;

FIG. 6 shows the results of field resistance identification of transgenic plants overexpressing the coding regions of SBR11 and SBR 11;

FIG. 7 shows the results of identifying the greenhouse resistance of transgenic plants overexpressing the coding regions of SBR11 and SBR 11;

FIG. 8 shows the greenhouse resistance identification results of transgenic plants transformed into the isogenic line NIL driven by the Ubiquitin promoter after the sites 64, 309 and 498 in SBR11 are changed respectively;

FIG. 9 shows the detection of dCAPS molecular markers at positions 64 and 309 of SBR11 in 8 cultivars, SNP190 at position 64 and SNP926 at position 309.

Detailed Description

The invention is further described below with reference to the accompanying drawings. The following examples are only for illustrating the technical solutions of the present invention more clearly, and the protection scope of the present invention is not limited thereby.

Example 1 verification of candidate genes

In the implementation, an anti-sheath blight candidate gene SBR11 is detected on LE chromosome 11 by using an isogenic line constructed by LE and TQ. In order to verify the function of the gene, the invention constructs SBR11 overexpression (SBR 11 OE) and an RNAi vector (SBR 11 RNAi), and the SBR11-OE vector is transferred into an infection near isogenic line NIL-SBR11 ^TQ (NIL), SBR11RNAi vector was transferred into LE. The results of field resistance identification show that compared with the control, the resistance of the SBR11OE transgenic line to banded sclerotial blight is obviously increased (figure 1), and the resistance of the SBR11RNAi transgenic line to banded sclerotial blight is obviously reduced (figure 2). The full-length gene of SBR No. 11 was cloned from LE and transferred into the susceptible near isogenic line NIL, together with the empty load control (# 8). The 8 independent transformed lines obtained (containing 1 empty control) were characterized for inoculation with Rhizoctonia solani. The results show (fig. 3) that the resistance of 4 lines was significantly higher than that of the susceptible control NIL and the empty control, and that the phenotype of 2 of these lines (# 2 and # 4) was essentially comparable to that of the wild-type control LE carrying SBR11. The results all prove that SBR11 positively regulates the resistance of rice to sheath blight.

Example 2 SBR11 functional analysis

The disease resistance allele of SBR11 (SBR 11) is from LE, and the disease susceptibility allele (SBR 11) is carried by indica variety TQ at this locus. Sequencing results showed (FIG. 4) that SBR No. 11 differed not only in 11 SNPs within 2kb upstream of ATG but also in 5 mutations in the coding region between the anti-and susceptible allele donor parents, with sense mutations at 190,926 and 1492. The induced expression level of SBR11 after the infection of rhizoctonia solani is obviously higher than that of SBR11 (figure 5). In order to verify the gene function of SBR11, the coding regions of two alleles of SBR11 and SBR11 are respectively driven by an overexpression promoter Ubiquitin and transferred into a susceptible near isogenic line NIL. The transgenic lines were subjected to the isolation of Rhizoctonia solani in greenhouse inoculation (FIG. 6) and identification of field inoculation (FIG. 7). The results show that the resistance of transgenic plants overexpressing the SBR11 coding region was significantly increased compared to the control, whereas the resistance of transgenic plants overexpressing the SBR11 coding region was not significantly changed. It was shown that the functional difference between SBR11 and SBR11 is mainly due to coding region variation.

Due to the difference of 3 amino acids in LE and TQ coding regions, the present invention amplified the DNA sequence of SBR No. 190,926,1492 with the changed bases, i.e. 64,309,498 amino acids were changed to TQ type respectively, and the specific amino acid changes are shown in Table 1. Driven by a Ubiquitin promoter, the gene is transferred into an isogenic line NIL of an infection by an agrobacterium-mediated method. The greenhouse inoculation identification result shows that the resistance of the NIL-SBR11OE transgenic plant to the sheath blight is obviously higher than that of the wild type NIL, when the amino acid at the 64 th position is changed from C to S, or the amino acid at the 309 th position is changed from S to N, the resistance of the transgenic material is obviously reduced, and the resistance of the amino acid at the 498 th position to the sheath blight of rice is not influenced by K or E. Therefore, it is presumed that amino acids 64 and 309 affect the resistance of SBR No. 11 to sheath blight.

TABLE 1 amino acid changes

	64	309	498
				TQ	Serine S	Aspartic acid N	Glutamic acid E
LE	Cysteine C	Serine S	Lysine K

Example 3 development of molecular markers

The invention develops dCAPS markers for gene selection aiming at SNP190 and SNP926 base variation, and the specific information of the two dCAPS markers is as follows:

SNP190: the molecular marker polymorphism is T/A at 4851244 th base of rice chromosome 11, namely 190 th base of SBR11 gene, the sequence of 176bp in front of and behind the SNP locus is shown as SEQ ID No.3, and the 154bp base is the SNP locus 190; the base of the SBR11 disease-resistant allele at the site is T and can not be identified by restriction enzyme PvuII, and the size of the amplified fragment is 176bp. The base of the disease allele sbr11 at the site is A, which is recognized by restriction enzyme PvuII, and 21bp fragments are cut off, leaving 155bp fragments.

SNP926: the base located on 4851980 th chromosome of rice No. 11, namely 926 th base of SBR11 gene, the molecular marker polymorphism is G/A, the sequence of 262bp before and after the SNP locus is shown as SEQ ID No.4, the 125 th base is SNP locus 926; the base of the SBR11 disease-resistant allele at the site is G, can be recognized by restriction enzyme DdeI and is cut into fragments of 124bp and 140 bp; the base of the disease allele sbr11 at this site is A, which is not recognized by the restriction enzyme DdeI, and is still 262bp.

Only when the SNP160 and the SNP926 are all of the sheath blight resistant homozygous genotypes, the sample to be detected can be judged to be the sheath blight resistant genotype. If any site is consistent with the susceptible genotype, the genotype of the sample to be detected is directly judged to be the susceptible sheath blight genotype.

The Primer for detecting the SNP site 190 is Primer190F and Primer190R, the sequence of the Primer190F is shown in SEQ.ID.NO.5, the sequence of the Primer190R is shown in SEQ.ID.NO.6, and the used restriction endonuclease is PvuII;

primers 926F and 926R are used as primers for detecting SNP position 926, the sequence of Primer926F is shown in seq.id.no.7, the sequence of Primer926R is shown in seq.id.no.8, and the restriction endonuclease used is DdeI.

Example 4 application of molecular markers

(1) Extracting the genomic DNA of rice to be detected

And 8 parts of rice samples to be detected, extracting rice genome DNA, and detecting the DNA concentration to ensure that A260/280 is between 1.8 and 2.0.

(2) And (3) PCR amplification:

PCR System (20 ul):

20ng of DNA template;

f primer (10 uM) is 0.5ul;

r primer (10 uM) is 0.5ul;

dNTP(2mM):2ul；

Mg ²⁺ (2.5mM):1.2ul；

10x PCR buffer:2ul；

0.2ul of Taq enzyme;

ddH ₂ o, complement to 20ul.

PCR amplification procedure:

pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 30s, extension at 72 ℃ for 30s, and after 38 cycles, keeping at 72 ℃ for 10min, and storing at 4 ℃ for detection.

(3) Enzyme digestion:

the enzyme digestion system is 10ul in total:

5ul of PCR product, 10xbuffer, endonuclease 0.3ul, ddH ₂ O is added to 10ul.

(4) 8 portions of rice samples to be tested and 2 portions of controls (LE and TQ) were subjected to PCR amplification and detected in 4% agarose gel electrophoresis after enzyme digestion.

SNP190: if a 176bp characteristic band appears in the electrophoresis result, the base of the rice to be detected at the SNP190 position is consistent with the SBR11 disease-resistant allele (namely the allele carried in LE); if a characteristic band of 155bp appears in the electrophoresis result, the base of the rice to be detected at the SNP190 is consistent with the SBR11 disease-sensitive allele SBR11 (namely the allele carried in the TQ);

SNP926: if two characteristic bands of 124bp and 140bp respectively appear in the electrophoresis result, the base of the rice to be detected at the SNP190 position is consistent with the SBR11 disease-resistant allele (namely the allele carried in LE); if a 262bp characteristic band appears, the base at SNP190 is consistent with SBR11 disease-sensitive allele SBR11 (i.e. the allele carried in TQ).

As a result, as shown in FIG. 9, in the SNP190 marker, the bands of samples Nos. 1, 2, 3, and 4 were 155bp, and it was found that the bases of samples Nos. 1, 2, 3, and 4 coincided with SBR11 disease-resistant allele SBR11 at SNP190, and the bands of samples Nos. 5, 6, 7, and 8 were 176bp, and it was found that the bases of samples Nos. 5, 6, 7, and 8 coincided with SBR11 disease-resistant allele SBR11 at SNP 190.

In the SNP926 marker, a 262bp appears in samples No.1, 2, 3, 4, 5 and 7, so that the base of the samples No.1, 2, 3, 4, 5 and 7 at the SNP190 position is consistent with the SBR11 disease-resistant allele SBR11, and the base of the samples No.6 and 8 at the SNP190 position is consistent with the SBR11 disease-resistant allele SBR11 by two characteristic bands of 124bp and 140bp respectively.

In conclusion, the samples No.6 and 8 contain SBR11 disease resistance allele SBR11, and the samples No.1, 2, 3, 4, 5 and 7 contain SBR11 susceptible allele SBR11.

The SNP190 and SNP926 markers have polymorphism in the varieties, clear band types, obvious polymorphism and moderate amplified fragment size, which indicates that the developed markers can successfully detect the genetic variation of SBR11 in different varieties of rice.

The method can quickly predict the resistance of the rice plant to the sheath blight disease and accelerate the selection progress of the sheath blight resistant rice material.

The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and variations can be made without departing from the technical principle of the present invention, and these modifications and variations should also be regarded as the protection scope of the present invention.

Sequence listing

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Claims

1. Used for detecting rice sheath blight resistance geneSBR11The primer composition of (1), which is characterized by comprising a primer set for detecting SNP site 190 and for detecting SNP site 926; primers for detecting the SNP site 190 are Primer190F and Primer190R, wherein the sequence of the Primer190F is shown as SEQ ID NO.5, and the sequence of the Primer190R is shown as SEQ ID NO. 6; the primers for detecting the SNP site 926 are Primer926F and Primer926R, wherein the sequence of the Primer926F is shown as SEQ ID NO.7, and the sequence of the Primer926R is shown as SEQ ID NO. 8.

2. Application of the primer composition of claim 1 in preparation of gene for detecting rice sheath blight resistanceSBR11The kit of (1).

3. Use according to claim 2, characterized in that: the application method of the kit comprises the following steps:

carrying out PCR amplification on sample genome DNA by primers with nucleotide sequences shown as SEQ ID NO.5 and SEQ ID NO.6, carrying out enzyme digestion on the PCR amplification product by using restriction endonuclease PvuII, and detecting the enzyme digestion product in agarose gel electrophoresis with the concentration of 4%; if a 176bp characteristic band appears in the electrophoresis result, the base of the rice to be detected at the SNP190 position andSBR11disease resistance allelesSBR11The consistency is achieved; if a characteristic band of 155bp appears in the electrophoresis result, the base of the rice to be detected at the SNP190 position andSBR11disease-sensitive allelessbr11The consistency is achieved;

carrying out PCR amplification on sample genome DNA by primers with nucleotide sequences shown as SEQ ID NO.7 and SEQ ID NO.8, carrying out enzyme digestion on the PCR amplification product by using restriction enzyme DdeI, and detecting the enzyme digestion product in agarose gel electrophoresis with the concentration of 4%; if two characteristic bands of 124bp and 140bp respectively appear in the electrophoresis result, the base of the rice to be detected at the SNP926 andSBR11disease resistance allelesSBR11The consistency is achieved; if a 262bp characteristic band appears, the base of the rice to be detected at the SNP190 position andSBR11disease-sensitive allelessbr11The consistency is achieved;

only the genome of the sampleBoth SNP190 and SNP926 are involvedSBR11Disease resistance allelesSBR11If they are consistent, the sample can be judged to containSBR11Disease resistance allelesSBR11Otherwise the sample containsSBR11Disease-sensitive allelessbr11。